Mechanism-Guided Synergistic Engineering of Substrate Access and Catalytic Microenvironment in Oxidosqualene Cyclase for Enhanced Amyrin Biosynthesis.

Oxidosqualene cyclases (OSCs) catalyze the cyclization of 2,3-oxidosqualene into diverse triterpenoids, yet their intrinsically low catalytic efficiency restricts biosynthetic productivity. Here, we establish a mechanism-guided synergistic engineering strategy that extends beyond conventional active-site engineering by integrating distal substrate access regulation with catalytic microenvironment optimization to enhance the catalytic performance of CrAS from Catharanthus roseus. Structural modeling and mechanistic analyses revealed a conserved catalytic framework involving carbocation-mediated polycyclization and identified a surface-exposed constriction region that regulates substrate access. Guided by these insights, distal surface engineering of the constriction region was synergistically combined with active pocket optimization. The resulting combinatorial mutant, M3 (L323A/T327K/N565I), exhibited a 95.2% increase in catalytic efficiency and enhanced α-amyrin and β-amyrin by 53.2% and 49.7%, reaching 158 mg/L and 63 mg/L, respectively. Multi-scale analyses combining molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the enhanced catalytic performance is attributable to increased flexibility of the substrate access pathway, reinforced electrostatic and cation-π interactions, and reduced reaction energy barriers. Notably, distal mutation T327K improved substrate ingress through dynamic modulation of the protein surface, while N565I optimized the catalytic microenvironment by enhancing hydrophobic packing and stabilizing key intermediates. Overall, our findings establish a generalizable framework for engineering complex cyclases and provide a foundation for the sustainable microbial production of high-value triterpenoids.